Abstract
We recently showed that inter-keratin disulfide bonding plays an important role in the assembly, organization, and dynamics of keratin intermediate filaments in skin keratinocytes. In particular, cysteine 367 located in the central α-helical rod domain of keratin 14 is necessary for the formation of a stable perinuclear network of keratin filaments (with type II partner keratin 5) in skin keratinocytes analyzed by static and live cell imaging. Here, we show that two additional cysteine residues located in the non-helical head domain of K14, Cys-4 and Cys-40, also participate in inter-keratin disulfide bonding and tandemly play a key role complementary to that of Cys-367 in the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in primary culture. Analysis of K14 variants with single or multiple substitutions of cysteine residues points to a spatial and temporal hierarchy in how Cys-4/Cys-40 and Cys-367 regulate keratin assembly in vitro and filament dynamics in live keratinocytes in culture. Our findings substantiate the importance and complexity of a novel determinant, namely inter-keratin disulfide bonding, for the regulation of several aspects of keratin filaments in surface epithelia.
Highlights
The functional importance of inter-keratin disulfide bonding is underappreciated in epithelial biology
We recently showed that inter-keratin disulfide bonding plays an important role in the assembly, organization, and dynamics of keratin intermediate filaments in skin keratinocytes
(16), we showed that replacing Cys with Ala at position 367 (K14C367A variant) decreased but did not eliminate disulfide-bonded K14 species in transfected 308 mouse keratinocytes, suggesting that another cysteine(s) in the K14 head is involved in intermolecular disulfide bonding
Summary
The functional importance of inter-keratin disulfide bonding is underappreciated in epithelial biology. We show that two additional cysteine residues located in the non-helical head domain of K14, Cys-4 and Cys-40, participate in inter-keratin disulfide bonding and tandemly play a key role complementary to that of Cys-367 in the assembly, organization, and dynamics of keratin filaments in skin keratinocytes in primary culture. Disulfide Bonding and Keratin Filaments larly, residue Cys-367 in the central ␣-helical rod domain of human K14 mediates a trans-dimer homotypic disulfide bond in a crystal of the interacting K5/K14 rod domains as well as in epidermal keratinocytes in vivo [16] This cysteine and its associated disulfide bonds play a key role in the formation and/or maintenance of a perinuclear-concentrated network of keratin IFs and in the centripetal keratin filament cycle observed in epidermal keratinocytes in primary culture [17]. We present evidence that all three cysteine residues act in concert to foster the execution of a normal keratin cycle in live cultures of epidermal keratinocytes
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